JP2005529619A - How to make carotenoids - Google Patents

Abstract

The present invention is capable of producing carotenoids and uses a microorganism belonging to the genus Xanthophyllomyces (Phaffia) in the presence of a farnesyl pyrophosphate-derived steroid biosynthesis inhibitor. Relates to biological methods of producing carotenoids.

Description

  The present invention uses a microorganism that is capable of producing carotenoids and belongs to the genus Xanthophyllomyces (Phaffia), and produces steroids derived from farnesyl pyrophosphate (hereinafter referred to as FPP). It relates to biological methods for producing carotenoids in the presence of synthesis inhibitors.

  Over 600 different carotenoids have been disclosed from carotenogenic organisms found in bacteria, yeast, fungi, and plants. Currently, only two of them, β-carotene and astaxanthin, are produced commercially in microorganisms and used in the food and feed industry. Since these carotenoids have strong antioxidant properties, they are industrially important as natural pigments and as functional substances for human health. In addition, astaxanthin colors fish in a unique orange-red color that contributes to appeal to consumers, so there is a demand for astaxanthin as a colorant from a commercial standpoint, especially in the fish farming industry such as salmon farming. Has increased.

  Other carotenoids such as lycopene, diaxanthin, canthaxanthin, and β-cryptoxanthin are also industrially important as natural pigments and antioxidants. Lycopene has been shown to efficiently quench singlet oxygen in experiments performed in vitro. Lycopene inhibits lipid peroxidation, but serum levels of this carcinoid are inversely proportional to cancer risk in the pancreas and cervix. The red color of fruits and vegetables like tomatoes, pink grapefruit, red grape skin, watermelon, and red guava is due to lycopene. Other food sources include papaya and apricot. Diaxanthin is a yellow carotenoid and an oxidized hydroxy derivative of β-carotene. Diaxanthin is abundant in seeds of spinach and corn, as well as many other plants. This is a strong antioxidant and is also found in the retina. It is widely believed that diaxanthin acts as a filter to protect the eye from harmful blue light and to protect the eye against age-related macular degeneration. Canthaxanthin is a red carotenoid and is found in many plants and animals. It is used for coloring egg yolks, broilers and cultured trout, and also in foods and cosmetics where orange-red is required. Canthaxanthin also functions as a singlet oxygen quencher and free radical inactivator. β-cryptoxanthin is a yellow carotenoid found in orange, mango, papaya, pumpkin and many other fruits. β-cryptoxanthin is also recognized as a strong antioxidant useful for cancer prevention.

  Among the microorganisms that can produce significant amounts of carotenoids, Phaffia rhodozyma is one of the best known carotenoid producing strains. This yeast strain produces astaxanthin and is one of the few microorganisms currently used to provide astaxanthin in the food and feed industries. Recent taxonomic studies have revealed the sex cycle of Phaffia rhodozyma, and the telemorphic state is Xanthophyllomyces dendrorhous [Golubev, Yeast 11: 101-110 (1995) )]. In this specification, the well-recognized name, Phaffia Rhodzyma, is used.

  For example, engineered to obtain the ability to produce significant amounts of some carotenoids using an appropriate host strain such as E. coli, Saccharomyces cerevisiae, or Candida utilis. Construction of recombinant microorganisms [Misawa et al., J. Bacteriol. 172: 6704 (1990); Yamamoto et al., Biosci. Biotech. Biochem. 58: 1112 (1994); Miura et al., Biotechnol. Bioeng. 58: 306 (1998)] For example, strain improvements to obtain high astaxanthin producers from Phaffia rhodozyma [Johnson et al., Critical Reviews in Biotechnology 11: 297-326 (1991)], and, for example, fermentation for production of astaxanthin by Phaffia rhodozyma Several studies have been conducted to increase the level of carotenoid production by microorganisms, including improved methods to optimize the process [US Pat. No. 5,972,642]. US Pat. No. 5,356,809 disclosed that administration of antimycin or other major respiratory chain inhibitors to Phaffia rhodozyma cells promotes astaxanthin production. However, this is not a more efficient or convenient method. Clearly, there is still a need for a simple and efficient method to increase the yield of carotenoid production.

  One aspect of the present invention provides an organism for producing carotenoids, comprising culturing a microorganism capable of producing carotenoids in an aqueous nutrient medium under aerobic conditions in the presence of an FPP-derived sterol biosynthesis inhibitor. Method.

  For example, it is more preferable to use a highly carotenoid-producing microorganism such as those belonging to the genus Xanthophyllomyces (Fafia). Accordingly, a preferred example of the microorganism of the present invention is a microorganism belonging to the genus Xanthophyllomyces (Phafia). Preferred strains of Xanthophyllomyces (Fafia) of the present invention are from the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, USA, Xanthophilomyces (Fafia) ATCC 96594 (Budapest). (According to the Convention, it was redeposited as accession number ATCC74438 on April 8, 1988).

  Accordingly, one embodiment of the present invention is a xanthophyllomyces (phaphia) in the presence of a substrate for producing carotenoids and an FPP-derived sterol biosynthesis inhibitor, particularly in an aqueous nutrient medium under aerobic conditions. An organism for producing carotenoids, comprising culturing a microorganism belonging to the genus and capable of producing carotenoids, and isolating the obtained carotenoids from the cells of the microorganisms or from the culture broth Related to scientific methods.

  In particular, inhibiting the initial reaction of the sterol pathway catalyzed by squalene synthase would be more preferred for the biological methods of the invention.

  Inhibitors of squalene synthase (also referred to in the technical field as squalene synthetase) have been reported to reduce intracellular sterol levels. Squalene synthase is an enzyme that catalyzes the first step involved in sterol biosynthesis. Two molecules of FPP are concentrated to form squalene.

  Accordingly, a further aspect of the invention is a biological method for producing carotenoids comprising culturing a microorganism capable of producing carotenoids in the presence of squalene synthase.

  Several types of squalene synthase inhibitors have been reported [Biller et al., Current Pharmaceutical Design 2: 1-40 (1996)]. Three broad groups of squalene synthase inhibitors have been reported, squalene synthase inhibitors based on ammonium ions, FPP mimetics containing phosphorus, and inhibitors based on carboxylic acids. Any type of squalene synthase inhibitor is preferred for the present invention. One preferred example of such a squalene synthetase inhibitor is an ammonium ion-based squalene synthetase inhibitor.

  Furthermore, one of the preferred examples of squalene synthetase inhibitors based on ammonium ions is a phenoxypropylamine type squalene synthetase inhibitor [Brown et al., Journal of Medicinal Chemistry 38: 4157-4160 (1995)]. For example, [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-methyl-N-isopropyl -3- (4-acetamido-2-allylphenoxy) propylamine, N-cyclopentyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-cyclobutyl-3- (4-acetamido-2-allylphenoxy) ) Propylamine, N-isopropyl-3- (2-allyl-4-butyramidephenoxy) propylamine, N-isopropyl-3- (4-acetamido-2-chlorophenoxy) propylamine, N-isopropyl-3- ( 4-acetamido-2-propylphenoxy) propylamine, and N-isopropyl-3- (4-acetamido-2-allylphenoxy) -1-methylpropylamine, or biologically acceptable salts thereof And many phenos Shi propylamine type squalene synthase inhibitors are known (Brown et al., Supra).

  Preferred squalene synthase inhibitors used in this example are [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, N-isopropyl-3- (4-acetamido-2-allyl) Phenoxy) propylamine, and N-methyl-N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine.

  Molasses, sucrose, or glucose and corn steep liquor, yeast extract, diammonium sulfate, ammonium phosphate as a carbohydrate source for cell growth and also as a substrate for carotenoid production Of nitrogen sources such as ammonium hydroxide or urea, phosphorus sources such as ammonium phosphate and phosphoric acid, and added micronutrients or inorganic salts such as magnesium sulfate, zinc sulfate, and biotin or desthiobiotin Carotenoids are usually produced by culturing carotenogenic microorganisms in a medium containing macronutrients and micronutrients appropriate for the cells.

  Preferred culture conditions are pH 4-8, temperature range 15 ° C-26 ° C, 24 hours-500 hours. Further preferable culture conditions are a pH range of 5 to 7 and a temperature range of 18 ° C. to 22 ° C. for 48 hours to 350 hours.

  In culture, aeration and agitation usually give favorable results for carotenoid production.

  In the present invention, an FPP-derived sterol biosynthesis inhibitor is added to the medium. The concentration of the inhibitor is suitably varied based on the type of inhibitor and microorganism used for carotenoid production, for example within a range of concentrations that reduce cell growth by less than 50% in carotenoid production conditions . A more preferred concentration of inhibitor may be in a concentration range that reduces cell growth by less than 30%.

  In the present invention, the FPP-derived sterol biosynthesis inhibitor may be added to the medium at any time during the culture.

  Carotenoids produced by culturing carotenoid-producing microorganisms using the method of the present invention can be prepared by methods known in the art [eg, Carotenoids Vol IA: Isolation and Analysis, Britton et al., Birkhauser Verlag, Basel (1995). Can be isolated from the medium if secreted into the medium or from the cells of the microorganism and, if necessary, separated from other carotenoids that may be present if one specific carotenoid is desired. Is done.

  Carotenoids made according to the present invention can be used in a method for the preparation of food or feed. Those skilled in the art are familiar with such methods. Such compound foods or feeds can further comprise additives or ingredients commonly used for such purposes and known in the art.

  The invention is further illustrated by the following examples, which do not limit the scope of the invention.

  The squalene synthase inhibitor used in this example is [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, one of a series of phenoxypropylamine type inhibitors. . This compound was synthesized according to the method described by Brown et al. That is, 3-allyl-biphenyl-4-ol was reacted with allyl bromide and potassium carbonate in butan-2-one and by thermal rearrangement to give biphenyl-4-ol (product code H7751, Sigma, USA). Prepared from. 3-Allyl-biphenyl-4-ol reacts with 1,3-dibromopropane and potassium carbonate in butan-2-one, followed by reaction with isopropylamine in 2-propanol to give [3- (3 Yielded -allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine.

Example 1: Effect of addition of a squalene synthase inhibitor on cell growth of Phaffia rhodozyma Phaffia rhodozyma ATCC 96594 was inoculated into YPD medium (DIFCO, Detroit, USA, 10 mL in a test tube) for 2 days at 20 ° C. Cultured with shaking. 0.5 mL cultures with 30 g / L glucose and 0 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL and Inoculate fresh YPD medium (10 mL in a test tube) containing 20.0 μg / mL [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine and shake at 20 ° C. for 5 days Cultured.

  Aliquots of the culture were taken from time to time during culture and absorbance at 660 nm was measured using a UV-1200 photometer (Shimadzu Corp., Kyoto, Japan) for analysis of cell growth. The results are shown in Table 1.

  When the concentration of the squalene synthase inhibitor was 2.0 μg / mL or less, cell growth was not affected. It was observed that 5.0 μg / mL inhibitor inhibited cell growth by about 23% on the second day.

Example 2: Effect of addition of squalene synthase inhibitor on astaxanthin produced by Phaffia rhodozyma Phaffia rhodozyma ATCC 96594 was inoculated into YPD medium as in Example 1 and cultured at 20 ° C for 2 days with shaking. 2.5 mL of culture was added to 22 g / L of glucose and 0 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL and 5.0 μg / mL of [3- (3-allyl-biphenyl- Fresh YPD medium (50 mL in flask) containing 4-yloxy) -propyl] -isopropyl-amine was inoculated and cultured with shaking at 20 ° C. for 7 days. On the second day of culture, 50 g / L glucose was added to the culture. Inhibitor addition during the culture was similarly tested. Aliquots of the culture were taken on days 4 and 7 of the culture, and the absorbance at 660 nm (by using the same method described in Example 1) and the astaxanthin content in the culture were measured. .

For analysis of astaxanthin content, extract the carotenoids from Phaffia rhodozyma cells by mixing the collected broth with a solvent mixture (ethyl alcohol, hexane, and ethyl acetate) and shaking vigorously with glass beads did. After extraction, the disrupted cells and glass beads were removed by centrifugation, and the resulting supernatant was analyzed for the amount of astaxanthin by HPLC. The HPLC conditions used are as follows.
HPLC column: Chrompack Lichrosorb si-60 (4.6 mm, 250 mm)
Temperature: Room temperature Eluent: Acetone / hexane (18/82) was added to 1 ml / L water for elution Injection volume: 10 μl
Flow rate: 2.0 ml / min Detection: UV at 450 nm

  A reference sample of astaxanthin was obtained from Hoffman La-Roche (Basel, Switzerland). The results are shown in Table 2.

^＊阻害剤の非存在下、7日目のアスタキサンチン含量を100として計算された相対値
^＊＊培養は阻害剤の非存在下で開始し、5.0μg/mLの阻害剤を、培養2日目に加えた

^* Relative value calculated as 100 astaxanthin content on day 7 in the absence of inhibitor
^** Culture started in the absence of inhibitor and 5.0 μg / mL inhibitor was added on day 2 of culture

  Astaxanthin production increased in all conditions tested. The best results were obtained when the inhibitor was added on the second day of culture, but addition of the inhibitor from the beginning of the culture was also effective for astaxanthin production.

Claims (12)

  A biological method for producing carotenoids comprising culturing a microorganism capable of producing carotenoids in an aqueous nutrient medium under aerobic conditions in the presence of a sterol biosynthesis inhibitor derived from farnesyl pyrophosphate.   In the presence of a sterol biosynthesis inhibitor derived from farnesyl pyrophosphate, a substrate for the production of carotenoids in an aqueous nutrient medium under aerobic conditions, the genus Xanthophyllomyces (Phaffia) A biological method for producing a carotenoid comprising the steps of culturing a microorganism belonging to and capable of producing a carotenoid and isolating a carotenoid obtained from a cell of the microorganism or from a cultured broth.   3. The method of claim 2, wherein the microorganism is Xanthophyllomyces dendrorhous (Phaffia rhodozyma) ATCC 96594.   3. The method according to claim 1, wherein the sterol biosynthesis inhibitor derived from farnesyl pyrophosphate is selected from the group consisting of squalene synthase inhibitors.   5. The method of claim 4, wherein the squalene synthase inhibitor is selected from the group consisting of ammonium ion based squalene synthase inhibitors.   6. The method of claim 5, wherein the ammonium ion based squalene synthetase inhibitor is selected from the group consisting of phenoxypropylamine type squalene synthetase inhibitors.   Phenoxypropylamine type squalene synthase inhibitors are [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, N-isopropyl-3- (4-acetamido-2-allylphenoxy) Propylamine, N-methyl-N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-cyclopentyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-cyclobutyl-3 -(4-Acetamido-2-allylphenoxy) propylamine, N-isopropyl-3- (2-allyl-4-butyramidephenoxy) propylamine, N-isopropyl-3- (4-acetamido-2-chlorophenoxy) Propylamine, N-isopropyl-3- (4-acetamido-2-propylphenoxy) propylamine, and N-isopropyl-3- (4-acetamido-2-allylphenoxy) -1-methylpropylamine, and Things histologically selected from acceptable group consisting of salts The method of claim 6 wherein.   The phenoxypropylamine type squalene synthetase inhibitor is [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine or a biologically acceptable salt thereof, N-isopropyl-3- 8. The method according to claim 7, which is (4-acetamido-2-allylphenoxy) propylamine or N-methyl-N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine.   3. The method of claim 1 or 2, wherein the concentration of the inhibitor is within a range of concentrations that reduce cell growth by less than 50% under carotenoid production conditions.   10. The method of claim 9, wherein the concentration of the inhibitor is within a range of concentrations that reduce cell growth by less than 30% under carotenoid production conditions.   The method according to claim 1 or 2, wherein the culturing is performed at a pH range of 4 to 8 and at a temperature range of 15 ° C to 26 ° C for 24 hours to 500 hours.   12. The method according to claim 11, wherein the culture is carried out at a pH range of 5 to 7 and at a temperature range of 18 ° C. to 22 ° C. for 48 hours to 350 hours.